Macrophages produce reactive oxygen species (ROS) and other antimicrobial substances to kill bacteria. Salmonella Typhimurium is a major food-borne pathogen capable of surviving within macrophages. SodCI, the periplasmic superoxide dismutase, directly and specifically protects Salmonella against superoxide, the primary phagocytic ROS. However, the mechanism by which phagocytic superoxide damages bacteria is unknown. Dogma states that bacterial DNA is a primary target of oxidative damage in the macrophage, consistent with known effects of ROS produced endogenously in the bacterial cytoplasm. But we have shown that, contrary to dogma, the targets of the phagocytic oxidative burst are not the bacterial DNA or other cytoplasmic molecules. Rather, phagocytic superoxide damages an extracytoplasmic bacterial target. Our goal is to understand the physiological basis of bacterial sensitivity to phagocytic superoxide and the mechanisms by which pathogenic bacteria protect against this innate immune response. Novel genetic and biochemical approaches are used to identify and study the bacterial targets of phagocytic superoxide. A variation on a high-throughput transposon mutagenesis technique that we call differential TnSeq was used to identify genes that show synthetic, epistatic, or suppressive genetic interaction with sodCI. The ytfL gene was identified as being epistatic to sodCI. Genes with the same phenotypic profile as ytfL are significantly enriched in genes that also show genetic interactions with sodCI. These data lead us to hypothesize that YtfL and the products of these related genes comprise a complex or system that is sensitive to exogenous superoxide, and that this system is normally protected by SodCI. The identified genes will be further characterized using in vivo and in vitro assays. To facilitate these studies, we have developed a novel in vitro device that, for the first time, allows the production of superoxide in the laboratoy at levels that mimic those produced in the phagocyte. These innovative approaches, along with the co-investigators' combined expertise in bacterial genetics, pathogenesis, physiology, and biochemistry of oxidative stress, make us uniquely qualified to address this fundamental aspect of innate immunity. Completion of the specific aims will increase our ability to prevent and treat, not only Salmonella infection, but also diseases caused by other pathogens.